System and method for processing samples

ABSTRACT

A system and method for processing samples. The system can include a loading chamber, a detection chamber positioned in fluid communication with the loading chamber, and a fluid path defined at least partially by the loading chamber and the detection chamber. The system can further include a filter positioned such that at least one of its inlet and its outlet is positioned in the fluid path. The method can include positioning a sample in the loading chamber, filtering the sample in the fluid path to form a concentrated sample and a filtrate, removing the filtrate from the fluid path at a location upstream of the detection chamber, moving at least a portion of the concentrated sample in the fluid path to the detection chamber, and analyzing at least a portion of the concentrated sample in the detection chamber for an analyte of interest.

FIELD

The present disclosure generally relates to a system and method forprocessing samples, and particularly, for processing liquid samples, andmore particularly, for processing dilute liquid samples.

BACKGROUND

Testing aqueous samples for the presence of microorganisms (e.g.,bacteria, viruses, fungi, spores, etc.) and/or other analytes ofinterest (e.g., toxins, allergens, hormones, etc.) can be important in avariety of applications, including food and water safety, infectiousdisease diagnostics, and environmental surveillance. For example,comestible samples, such as foods, beverages, and/or public waterconsumed by the general population may contain or acquire microorganismsor other analytes, which can flourish or grow as a function of theenvironment in which they are located. This growth may lead to theproliferation of pathogenic organisms, which may produce toxins ormultiply to infective doses. By way of further example, a variety ofanalytical methods can be performed on samples of non-comestible samples(e.g., groundwater, urine, etc.) to determine if a sample contains aparticular analyte. For example, groundwater can be tested for amicroorganism or a chemical toxin; and urine can be tested for a varietyof diagnostic indicators to enable a diagnosis (e.g., diabetes,pregnancy, etc.).

SUMMARY

One aspect of the present disclosure provides a method for processingsamples. The method can include providing a loading chamber, providing adetection chamber positioned in fluid communication with the loadingchamber, and providing a fluid path defined at least partially by theloading chamber and the detection chamber. The method can furtherinclude positioning a sample in the loading chamber, filtering thesample in the fluid path to form a concentrated sample and a filtrate,and removing the filtrate from the fluid path at a location upstream ofthe detection chamber. The method can further include moving at least aportion of the concentrated sample in the fluid path to the detectionchamber, and analyzing the at least a portion of the concentrated samplein the detection chamber for an analyte of interest.

Another aspect of the present disclosure provides a system forprocessing samples. The system can include a loading chamber adapted toreceive a sample, a detection chamber positioned in fluid communicationwith the loading chamber, and a fluid path defined at least partially bythe loading chamber and the detection chamber. The system can furtherinclude a filter having an inlet and an outlet, the filter positionedsuch that at least one of the inlet and the outlet is positioned in thefluid path. The filter can be adapted to filter the sample to form aconcentrated sample and a filtrate. The system can further include afiltrate outlet positioned such that the filtrate is removed from thefluid path at a location upstream of the detection chamber.

Other features and aspects of the present disclosure will becomeapparent by consideration of the detailed description and accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a front perspective view of a sample processing systemaccording to one embodiment of the present disclosure, the sampleprocessing system shown coupled to a vacuum source.

FIG. 2 a partial rear plan view of the sample processing system of FIG.1.

FIG. 3 is a partial side cross-sectional view the sample processingsystem of FIGS. 1 and 2, taken along line 3-3 in FIG. 1, with portionsremoved for clarity.

FIG. 4 is a front perspective view of a sample processing systemaccording to another embodiment of the present disclosure.

FIG. 5 is a partial side cross-sectional view of the sample processingsystem of FIG. 4, taken along line 5-5 in FIG. 4.

FIG. 6 is a partial side cross-sectional view of a sample processingsystem according to another embodiment of the present disclosure.

FIGS. 7-10 are photographs of assay results according to Example 1.

FIGS. 11-12 are photographs of assay results according to Example 2.

DETAILED DESCRIPTION

Before any embodiments of the present disclosure are explained indetail, it is to be understood that the invention is not limited in itsapplication to the details of construction and the arrangement ofcomponents set forth in the following description or illustrated in thefollowing drawings. The invention is capable of other embodiments and ofbeing practiced or of being carried out in various ways. Also, it is tobe understood that the phraseology and terminology used herein is forthe purpose of description and should not be regarded as limiting. Theuse of “including,” “comprising,” or “having” and variations thereofherein is meant to encompass the items listed thereafter and equivalentsthereof as well as additional items. Unless specified or limitedotherwise, the terms “connected” and “coupled” and variations thereofare used broadly and encompass both direct and indirect connections andcouplings. Further, “connected” and “coupled” are not restricted tophysical or mechanical connections or couplings. It is to be understoodthat other embodiments may be utilized, and structural or logicalchanges may be made without departing from the scope of the presentdisclosure. Furthermore, terms such as “front,” “rear,” “top,” “bottom,”and the like are only used to describe elements as they relate to oneanother, but are in no way meant to recite specific orientations of theapparatus, to indicate or imply necessary or required orientations ofthe apparatus, or to specify how the invention described herein will beused, mounted, displayed, or positioned in use.

In a variety of samples that are desired to be tested for one or moreanalytes of interest, the analyte(s) can be present in the sample at alow concentration and/or the sample volume can be large. Such situationscan require that the sample be concentrated in order to reach anappropriate concentration of an analyte of interest so as to achieve adetection threshold of an analytical technique. In some existing systemsand methods, membrane filtration can be employed to concentratelow-concentration samples, and can include capturing an analyte ofinterest onto microporous membranes for the purpose of identificationand/or enumeration. Such methods can include vacuum filtration of asample followed by sterile transfer of the membrane to another container(e.g., a Petri dish) containing the necessary nutrients that can thendiffuse through the pores in the membrane during incubation to allowbacterial colonies to form on the membrane. The concentration ofmicroorganisms in the original sample can then be determined by countingthe number of colonies present on the membrane. Such methods can requirenumerous steps, including preparation of growth containers,sterilization and set-up of the filtration apparatus, and steriletransfer of the membrane from the device to the growth media. Inaddition, the procedure is limited to use on solid or semi-solidnutrient formulations to limit excess diffusion of bacteria of interestand/or water-soluble indicator species. Other existing systems andmethods that include capturing cells on a support for further analysisor detection in one device do not allow for subsequent elution of theintact, captured cells or substantial removal of undesired filtrate fromthe device.

The present disclosure generally relates to a system and method forprocessing samples. In general, methods of the present disclosure caninclude concentrating samples to form a concentrated sample, moving atleast a portion of the concentrated sample in a fluid path to adetection chamber (e.g., without exposing the concentrated sample toambience), and analyzing the concentrated sample in the detectionchamber to determine at least one of the presence, quantity, and/orviability of an analyte of interest. Systems of the present disclosuregenerally include means for concentrating and assaying a sample in onesystem without exposing the sample to ambience.

The present disclosure can provide sample concentration andassaying/detection in a single system, which can allow large samplevolumes to be interrogated, can simplify the sample processing method,can reduce sample and environmental contamination, and can increaseassay sensitivity and accuracy by removing undesired portions of thesample from the assay. The sample processing method can be simplifiedand the contamination can be reduced at least partially because byallowing sample concentration and detection to be contained within asingle system of the present disclosure, a transfer step (e.g., of acollection membrane) into a second, separate device can be eliminated.In addition, in some embodiments of the present disclosure, where thesample is filtered in the system can be spatially separated from whereit is assayed, which can improve assay sensitivity and accuracy, atleast partially because undesired portions of the sample were removedfrom the assay.

The samples to be processed can be obtained in a variety of ways. Forexample, in some embodiments, the sample to be processed is itself aliquid sample, such as a dilute liquid sample and/or a dilute aqueoussample. In some embodiments, the sample can include the liquid resultingfrom washing or rinsing a source of interest (e.g., a surface, fomite,etc.) with a diluent. In some embodiments, the sample can include thefiltrate resulting from filtering a liquid composition resulting fromcombining a source of interest with an appropriate diluent. That is,large insoluble matter, such as various foods, fomites, or the like, canbe removed from a liquid composition in a first filtration step to formthe sample that will be processed using a sample processing system andmethod of the present disclosure.

The term “source” can be used to refer to a food or nonfood desired tobe tested for analytes. The source can be a solid, a liquid, asemi-solid, a gelatinous material, and combinations thereof. In someembodiments, the source can be provided by a substrate that was used,for example, to collect the source from a surface of interest. In someembodiments, the liquid composition can include the substrate, which canbe further broken apart (e.g., during an agitation or dissolutionprocess) to enhance retrieval of the source and any analyte of interest.The surface of interest can include at least a portion of a variety ofsurfaces, including, but not limited to, walls (including doors),floors, ceilings, drains, refrigeration systems, ducts (e.g., airducts),vents, toilet seats, handles, doorknobs, handrails, bedrails (e.g., in ahospital), countertops, tabletops, eating surfaces (e.g., trays, dishes,etc.), working surfaces, equipment surfaces, clothing, etc., andcombinations thereof. All or a portion of the source can be used toobtain a sample that is to be processed using the sample processingsystem and method of the present disclosure.

The term “food” is generally used to refer to a solid, liquid (e.g.,including, but not limited to, solutions, dispersions, emulsions,suspensions, etc., and combinations thereof) and/or semi-solidcomestible composition. Examples of foods include, but are not limitedto, meats, poultry, eggs, fish, seafood, vegetables, fruits, preparedfoods (e.g., soups, sauces, pastes), grain products (e.g., flour,cereals, breads), canned foods, milk, other dairy products (e.g.,cheese, yogurt, sour cream), fats, oils, desserts, condiments, spices,pastas, beverages, water, animal feed, other suitable comestiblematerials, and combinations thereof.

The term “nonfood” is generally used to refer to sources of interestthat do not fall within the definition of “food” and are generally notconsidered to be comestible. Examples of nonfood sources can include,but are not limited to, clinical samples, cell lysates, whole blood or aportion thereof (e.g., serum), other bodily fluids or secretions (e.g.,saliva, sweat, sebum, urine), feces, cells, tissues, organs, biopsies,plant materials, wood, soil, sediment, medicines, cosmetics, dietarysupplements (e.g., ginseng capsules), pharmaceuticals, fomites, othersuitable non-comestible materials, and combinations thereof.

The term “fomite” is generally used to refer to an inanimate object orsubstrate capable of carrying infectious organisms and/or transferringthem. Fomites can include, but are not limited to, cloths, mop heads,towels, sponges, wipes, eating utensils, coins, paper money, cellphones, clothing (including shoes), doorknobs, feminine products,diapers, etc., portions thereof, and combinations thereof.

The term “analyte” is generally used to refer to a substance to bedetected (e.g., by a laboratory or field test). A sample can be testedfor the presence, quantity and/or viability of particular analytes. Suchanalytes can be present within a source (e.g., on the interior), or onthe exterior (e.g., on the outer surface) of a source. Examples ofanalytes can include, but are not limited to, microorganisms,biomolecules, chemicals (e.g. pesticides, antibiotics), metal ions (e.g.mercury ions, heavy metal ions), metal-ion-containing complexes (e.g.,complexes comprising metal ions and organic ligands), and combinationsthereof.

A variety of testing methods can be used to identify, quantitate, and/orelucidate the viability of an analyte, including, but not limited to,microbiological assays, biochemical assays (e.g. immunoassay), or acombination thereof. Specific examples of testing methods that can beused include, but are not limited to, lateral flow assays, titration,thermal analysis, microscopy (e.g., light microscopy, fluorescentmicroscopy, immunofluorescent microscopy, scanning electron microscopy(SEM), transmission electron microscopy (TEM)), spectroscopy (e.g., massspectroscopy, nuclear magnetic resonance (NMR) spectroscopy, Ramanspectroscopy, infrared (IR) spectroscopy, x-ray spectroscopy, attenuatedtotal reflectance spectroscopy, Fourier transform spectroscopy,gamma-ray spectroscopy, etc.), spectrophotometry (e.g., absorbance,fluorescence, luminescence, etc.), chromatography (e.g., gaschromatography, liquid chromatography, ion-exchange chromatography,affinity chromatography, etc.), electrochemical analysis, genetictechniques (e.g., polymerase chain reaction (PCR), transcriptionmediated amplification (TMA), hybridization protection assay (HPA), DNAor RNA molecular recognition assays, etc.), adenosine triphosphate (ATP)detection assays, immunological assays (e.g., enzyme-linkedimmunosorbent assay (ELISA)), cytotoxicity assays, viral plaque assays,techniques for evaluating cytopathic effect, culture techniques such asthose that can be done using a growth medium (e.g., agar) and/or 3M™PETRIFILM™ plates (e.g., and imaged, quantified and/or interpreted usinga 3M™ PETRIFILM™ plate reader (3M Company, St. Paul, Minn.)), invasivecleavage reaction assays (e.g., INVADER® assay, available from ThirdWave Technologies, Madison, Wis., a wholly-owned subsidiary of Hologic,Inc., Bedford, Mass.), SMARTDNA™ assay (available from Investigen, Inc.,Hercules, Calif.), other suitable analyte testing methods, or acombination thereof.

The term “microorganism” is generally used to refer to any prokaryoticor eukaryotic microscopic organism, including without limitation, one ormore of bacteria (e.g., motile or vegetative, Gram positive or Gramnegative), viruses (e.g., Norovirus, Norwalk virus, Rotavirus,Adenovirus, DNA viruses, RNA viruses, enveloped, non-enveloped, humanimmunodeficiency virus (HIV), human Papillomavirus (HPV), etc.),bacterial spores or endospores, algae, fungi (e.g., yeast, filamentousfungi, fungal spores), prions, mycoplasmas, and protozoa. In some cases,the microorganisms of particular interest are those that are pathogenic,and the term “pathogen” is used to refer to any pathogenicmicroorganism. Examples of pathogens can include, but are not limitedto, members of the family Enterobacteriaceae, or members of the familyMicrococaceae, or the genera Staphylococcus spp., Streptococcus, spp.,Pseudomonas spp., Enterococcus spp., Salmonella spp., Legionella spp.,Shigella spp., Yersinia spp., Enterobacter spp., Escherichia spp.,Bacillus spp., Listeria spp., Campylobacter spp., Acinetobacter spp.,Vibrio spp., Clostridium spp., and Corynebacterium spp. Particularexamples of pathogens can include, but are not limited to, Escherichiacoli including enterohemorrhagic E. coli e.g., serotype O157:H7,O129:H11; Pseudomonas aeruginosa; Bacillus cereus; Bacillus anthracis;Salmonella enteritidis; Salmonella enterica serotype Typhimurium;Listeria monocytogenes; Clostridium botulinum; Clostridium perfringens;Staphylococcus aureus; methicillin-resistant Staphylococcus aureus;Campylobacter jejuni; Yersinia enterocolitica; Vibrio vulnificus;Clostridium difficile; vancomycin-resistant Enterococcus; andEnterobacter [Cronobacter] sakazakii. Environmental factors that mayaffect the growth of a microorganism can include the presence or absenceof nutrients, pH, moisture content, oxidation-reduction potential,antimicrobial compounds, temperature, atmospheric gas composition andbiological structures or barriers.

The term “biomolecule” is generally used to refer to a molecule, or aderivative thereof, that occurs in or is formed by an organism. Forexample, a biomolecule can include, but is not limited to, at least oneof an amino acid, a nucleic acid, a polypeptide, a protein, apolynucleotide, a lipid, a phospholipid, a saccharide, a polysaccharide,and combinations thereof. Specific examples of biomolecules can include,but are not limited to, a metabolite (e.g., staphylococcal enterotoxin),an allergen (e.g., peanut allergen(s), egg allergen(s), pollens, dustmites, molds, danders, or proteins inherent therein, etc.), a hormone, atoxin (e.g., Bacillus diarrheal toxin, aflatoxin, Clostridium difficiletoxin etc.), RNA (e.g., mRNA, total RNA, tRNA, etc.), DNA (e.g., plasmidDNA, plant DNA, etc.), a tagged protein, an antibody, an antigen, ATP,and combinations thereof.

The terms “soluble matter” and “insoluble matter” are generally used torefer to matter that is relatively soluble or insoluble in a givenmedium, under certain conditions. Specifically, under a given set ofconditions, “soluble matter” is matter that goes into solution and canbe dissolved in the solvent (e.g., diluent) of a system. “Insolublematter” is matter that, under a given set of conditions, does not gointo solution and is not dissolved in the solvent of a system. A sourcecan include soluble matter and insoluble matter (e.g., cell debris).Insoluble matter is sometimes referred to as particulate(s) or debrisand can include portions of the source material itself (i.e., frominternal portions or external portions (e.g., the outer surface) of thesource) or other source residue or debris resulting from an agitationprocess. The analyte of interest can be present in the soluble matter orthe insoluble matter.

The term “diluent” is generally used to refer to a liquid added to asource material to disperse, dissolve, suspend, emulsify, wash and/orrinse the source. In addition, a diluent can be added to a system of thepresent disclosure during one or more of concentration, elution, anddetection. In some embodiments, the diluent is a sterile liquid. In someembodiments, the diluent can include a variety of additives, including,but not limited to, surfactants, or other suitable additives that aid indispersing, dissolving, suspending or emulsifying the source forsubsequent analyte testing; rheological agents; antimicrobialneutralizers (e.g., that neutralize preservatives or other antimicrobialagents); enrichment or growth medium comprising nutrients (e.g., thatpromote selective growth of desired microorganism(s)) and/or growthinhibitors (e.g., that inhibit the growth of undesiredmicroorganism(s)); pH buffering agents; enzymes; indicator molecules(e.g. pH or oxidation/reduction indicators); spore germinants; an agentto neutralize sanitizers (e.g., sodium thiosulfate neutralization ofchlorine); an agent intended to promote bacterial resuscitation (e.g.,sodium pyruvate); stabilizing agents (e.g., that stabilize theanalyte(s) of interest, including solutes, such as sodium chloride,sucrose, etc.); or a combination thereof. In some embodiments, thediluent can include sterile water (e.g., sterile double-distilled water(ddH₂O)); one or more organic solvents to selectively dissolve,disperse, suspend, or emulsify the source; aqueous organic solvents, ora combination thereof. In some embodiments, the diluent is a sterilebuffered solution (e.g., Butterfield's Buffer, available from EdgeBiological, Memphis Term.). In some embodiments, the diluent is aselective or semi-selective nutrient formulation, such that the diluentmay be used in the selective or semi-selective growth of the desiredanalyte(s) (e.g., bacteria). In such embodiments, the diluent can beincubated with a source for a period of time (e.g., at a specifictemperature) to promote such growth and/or development of the desiredanalyte(s).

Examples of growth medium can include, but are not limited to, TrypticSoy Broth (TSB), Buffered Peptone Water (BPW), Universal Pre-enrichmentBroth (UPB), Listeria Enrichment Broth (LEB), Lactose Broth, Boltonbroth, or other general, non-selective, or mildly selective media knownto those of ordinary skill in the art. The growth medium can includenutrients that support the growth of more than one desired microorganism(i.e., analyte of interest).

Examples of growth inhibitors can include, but are not limited to, bilesalts, sodium deoxycholate, sodium selenite, sodium thiosulfate, sodiumnitrate, lithium chloride, potassium tellurite, sodium tetrathionate,sodium sulphacetamide, mandelic acid, selenite cysteine tetrathionate,sulphamethazine, brilliant green, malachite green oxalate, crystalviolet, Tergitol 4, sulphadiazine, amikacin, aztreonam, naladixic acid,acriflavine, polymyxin B, novobiocin, alafosfalin, organic and mineralacids, bacteriophages, dichloran rose bengal, chloramphenicol,chlortetracycline, certain concentrations of sodium chloride, sucroseand other solutes, and combinations thereof.

The term “agitate” and derivatives thereof is generally used to describethe process of giving motion to a material or an object (e.g., a sampleprocessing system of the present disclosure), for example, to break up,homogenize, mix, combine and/or blend various components/contents. Forexample, agitation can be used during one or more of sampleconcentration, elution, and detection. A variety of agitation methodscan be used, including, but not limited to, manual shaking, mechanicalshaking (e.g., linear shaking), sonic (e.g., ultrasonic) vibration,vortex stirring, mechanical stirring (e.g., by a magnetic stir bar, oranother agitating aid, such as ball bearings), manual compression (e.g.,manual beating, squeezing, kneading, pummeling, etc., and combinationsthereof), mechanical compression (e.g., mechanical beating, squeezing,kneading, pummeling, etc., and combinations thereof), and combinationsthereof.

The term “filtering” is generally used to refer to the process ofseparating matter by size, charge and/or function. For example,filtering can include separating soluble matter and a solvent (e.g.,diluent) from insoluble matter, or filtering can include separatingsoluble matter, a solvent and relatively small insoluble matter fromrelatively large insoluble matter. As a result, filtering can refer to(1) any pre-filtering steps that are employed to obtain a sample that isto be processed using the sample processing systems and methods of thepresent disclosure, (2) any filtering step that is employed toconcentrate the sample using the sample processing systems and methodsof the present disclosure, or (3) both (1) and (2). A variety offiltration methods can be used, including, but not limited to, passingthe liquid composition (e.g., comprising a source of interest, fromwhich a sample to processed can be obtained) through a filter, othersuitable filtration methods, and combinations thereof.

“Settling” is generally used to refer to the process of separatingmatter by density, for example, by allowing the more dense matter in theliquid composition (i.e., the matter having a higher density than thediluent and other matter in the mixture) to settle. Settling may occurby gravity or by centrifugation. The more dense matter can then beseparated from the less dense matter (and diluent) by aspirating theless dense (i.e., unsettled or floating) and diluent from the more densematter, decanting the less dense matter and diluent, or a combinationthereof. Pre-settling steps can be used in addition to or in lieu ofpre-filtering steps to obtain a sample that is to be processed using thesample processing systems and methods of the present disclosure.

A “filter” is generally used to describe a device used to separate thesoluble matter (or soluble matter and relatively small insoluble matter)and solvent from the insoluble matter (or relatively large insolublematter) in a liquid composition and/or to filter a sample during sampleconcentration. Examples of filters can include, but are not limited to,a woven or non-woven mesh (e.g., a wire mesh, a cloth mesh, a plasticmesh, etc.), a woven or non-woven polymeric web (e.g., comprisingpolymeric fibers laid down in a uniform or nonuniform process, which canbe calendered), a surface filter, a depth filter, a membrane (e.g., aceramic membrane (e.g., ceramic aluminum oxide membrane filtersavailable under the trade designation ANOPORE from Whatman Inc., FlorhamPark, N.J.), a polycarbonate membrane (e.g., track-etched polycarbonatemembrane filters available under the trade designation NUCLEOPORE fromWhatman, Inc.)), a polyester membrane (e.g., comprising track-etchedpolyester, etc.), a sieve, glass wool, a frit, filter paper, foam, etc.,and combinations thereof.

In some embodiments, the term “filtrate” is generally used to describethe liquid remaining after the insoluble matter (or at least therelatively large insoluble matter) has been separated or removed from aliquid composition. In some embodiments, the term “supernatant” isgenerally used to describe the liquid remaining after the more densematter has been separated or removed from a liquid composition. Such afiltrate and/or supernatant can form a sample to be processed by thesample processing systems and methods of the present disclosure.Alternatively, the term “filtrate” can be used to describe theundesirable portions of a sample that are removed during sampleconcentration of the sample processing systems and methods of thepresent disclosure.

Sample concentration can include filtering a sample to obtain aconcentrated sample, which can then be further processed andinterrogated. As mentioned above, filtering can refer to separatingmatter by size, charge and/or function. While the embodiments describedbelow and illustrated in FIGS. 1-6 can be used in a variety of sampleprocessing methods of the present disclosure, the embodimentsillustrated in FIGS. 1-5 are generally used to concentrate a sample ofinterest by size-filtration, and FIG. 6 is generally used to concentratea sample of interest by charge- and/or function-filtration.

FIGS. 1-3 illustrate a sample processing system 100 according to oneembodiment of the present disclosure. The sample processing system 100includes a loading chamber 102, a plurality of detection chambers 104,and a plurality of primary channels 106 positioned to fluidly couple theplurality of detection chambers 104 to the loading chamber 102. Thesample processing system 100 further includes a plurality of secondarychannels 108, and each secondary channel 108 is positioned to fluidlycouple one or more detection chambers 104 (i.e., one detection chamber104 in the embodiment illustrated in FIGS. 1-3) to the primary channel106.

The sample processing system 100 further includes a fluid path 110 thatis at least partially defined by one or more of the loading chamber 102,the plurality of detection chambers 104, the plurality of primarychannels 106, and the plurality of secondary channels 108. A sample canbe analyzed in the detection chambers 104 to elucidate at least one ofthe presence, quantity and/or viability of the analyte(s) of interest.

The sample processing system 100 can further include or be coupled to aport 107 via which a sample can be introduced into the loading chamber102. A valve can be positioned in fluid communication with the port 107(e.g., in the port 107 itself) to control the movement of a sample intothe loading chamber 102. In addition, the port 107 can be coupled to anupstream system or process via a connector 109. As mentioned above, thesample can be pre-filtered and/or pre-settled prior to being introducedinto the sample processing system 100. Such a “pre-filter” can bepositioned upstream of the connector 109, in the connector 109, in theport 107, and/or over an aperture 111 in the loading chamber 102 throughwhich the sample passes as the sample is introduced into the loadingchamber 102. In some embodiments, the port 107 includes a Luer lock tofacilitate controlling introduction of a sample into the loading chamber102 and/or connecting the port 107 to the connector 109 and/or any otherupstream system.

Pre-filtering and/or pre-settling a liquid composition upstream of thesample processing system 100 to form a sample that will be introducedinto the sample processing system 100 can be advantageous to enhance thepurity of the material passing through the sample processing system 100and enhance the capture of the analyte(s) of interest. In addition,pre-filtering and/or pre-settling can help avoid clogging the filters120 or other downstream portions of the sample processing system 100.However, pre-filtering and/or pre-settling the sample or an upstreamliquid composition is not necessary, and in some embodiments, the sampleis introduced directly into the sample processing system 100 withoutbeing pre-filtered and/or pre-settled.

As shown in FIGS. 1-3, the sample processing system 100 can include afirst major side 103 and a second major side 105. The first and secondmajor sides 103 and 105 can be manufactured in a variety of ways usingany suitable material or materials. Examples of suitable materialsinclude polymeric materials (e.g., polypropylene, polyester,polycarbonate, polyethylene, etc.), metals (e.g., metal foils), etc., orcombinations thereof. In some embodiments, one or more of the loadingchamber 102, the detection chambers 104, the primary channels 106 andthe secondary channels 108 can be formed in one side 103/105 of thesample processing system 100, while the opposite side 105/103 isprovided in a generally flat sheet-like configuration. For example, insome embodiments, one or more of the loading chamber 102, the detectionchambers 104, the primary channels 106, and the secondary channels 108can be formed in the first major side 103 in a polymeric sheet, forexample, that has been molded, vacuum-formed, thermoformed, or otherwiseprocessed. The second major side 105 can then be provided as, forexample, a sheet of metal foil, polymeric material (e.g., a polymericfilm), multi-layer composite, etc., or a combination thereof, that canbe coupled to the first major side 103. In some embodiments, thematerials can be selected for the first and second major sides 103 and105 that exhibit good water barrier properties.

As shown in FIGS. 1-3, the sample processing system 100 can furtherinclude one or more filters 120 positioned in the loading chamber 102.Each filter 120 can include an inlet 122 and an outlet 124, and eachfilter 120 can be positioned such that at least one of the inlet 122 andthe outlet 124 is positioned in the fluid path 110. For example, in theembodiment illustrated in FIGS. 1-3, the filter inlet 122 is positionedin the fluid path 110, and the outlet 124 is positioned such that anyfiltrate formed by the filter 120 is removed from the fluid path 110 viathe filter outlet 124. As a result, the filter 120 defines a filterfluid path 125 (see FIGS. 2 and 3). As such, the fluid path 110 can bereferred to as a “primary fluid path” of the system 100, and the filterfluid path 125 can be referred to as a “secondary fluid path,” and insome embodiments, the secondary fluid path 125 can be oriented at anangle with respect to the primary fluid path 110. For example, as shownin FIG. 3, the fluid path 110 is configured such that fluid can flow inthe fluid path 110 generally along a first direction D₁, and the filter120 is arranged such that fluid can flow through the filter 120 in thefilter fluid path 125 generally along a second direction D₂, and by wayof example only, the first direction D₁ and the second direction D₂ areshown as being oriented substantially perpendicularly with respect toone another, although other angles of orientation are possible. In someembodiments, as shown in FIG. 6, the filter fluid path 125 can beoriented in line with the fluid path 110 of the sample processing system100, which is described in greater detail below.

Each filter 120 can be adapted to form a concentrated sample and afiltrate. The sample processing system 100 can include an outlet 126,i.e., a filtrate outlet, for removing the filtrate of the sample, or theundesired portions of the sample, from the fluid path 110, andparticularly, for removing the filtrate from the fluid path 110 at alocation upstream of the detection chambers 104. In some embodiments, asshown in FIGS. 1-3 (and FIGS. 4-5, described below), the filter 120 ispositioned such that the filter outlet 124 is directed out of the fluidpath 110, and, as a result, the filtrate outlet 126 includes the filteroutlet 124. That is, in the embodiment in FIGS. 1-3, the filter 120 ispositioned such that portions of a sample flowing through the filter 120(i.e., the filtrate) will be directed out of the fluid path 110, andparticularly, away from the detection chambers 104.

In some embodiments, the filtrate outlet 126 can be in fluidcommunication with the fluid path 110 and positioned adjacent adownstream side of the filter 120 (but does not necessarily include thefilter outlet 124), such that the filtrate is removed from the fluidpath 110 upstream of the detection chambers 104. In some embodiments,the filtrate outlet 126 can be positioned in fluid communication withthe fluid path 110 at a location downstream of the filter 120 andupstream of the detection chambers 104 (e.g., as shown in the embodimentillustrated in FIG. 6 and described below). Whether the filtrate outlet126 includes the filter outlet 124, is adjacent the filter outlet 124,and/or is in fluid communication with the fluid path 110 at a locationdownstream of the filter 120, the filtrate outlet 126 can be positionedsuch that the filtrate can be removed from the fluid path 110 of thesample processing system 100 at a location upstream of the detectionchambers 104.

A sample can be filtered using the filters 120 by employing a pressuredifferential across each filter 120. That is, a pressure differentialcan be established between the upstream side (i.e., the inlet 122) andthe downstream side (i.e., the outlet 124) of each filter 120. In someembodiments, the pressure differential can be established by applyingpositive pressure (e.g., using an upstream manual pump, such as asyringe, bulb, or the like, a mechanical pump, or a combination thereof)to the upstream side of the filter 120 and/or by applying negativepressure to the downstream side of the filter 120. Furthermore, in someembodiments, a portion of the sample processing system 100 can bedeformable to create the pressure differential. For example, in someembodiments, the loading chamber 102 can be deformable to force thesample through the filters 120. In embodiments in which positivepressure is applied to an upstream side of the filter(s) 120, one ormore obstructions or seals can be moved into a position that obstructsand/or seals a downstream portion of the fluid path 110 to inhibit theunconcentrated sample from prematurely moving into the detectionchambers 104. Such obstructions and/or seals can be reversible, suchthat the fluid path 110 can be reopened when necessary.

In some embodiments, a vacuum source 130 can be coupled to the sampleprocessing system 100, such that the vacuum source 130 is in fluidcommunication with the fluid path 110 via the filtrate outlet 126. Inembodiments in which the filtrate outlet 126 includes or is in line withthe filter outlet 124, the vacuum source 130 can be coupled to thefilter outlet 124 to remove the filtrate of the sample of interest fromthe fluid path 110. The vacuum source 130 can include, but is notlimited to, a mechanical pump, a manual pump such as a syringe-plungercombination, etc., or a combination thereof that creates a reducedpressure. In embodiments employing negative pressure on the downstreamside of the filters 120, the entire fluid path 110 can be evacuated aswell, such that obstructions and/or seals do not need to be employed toinhibit the unconcentrated sample from prematurely moving into thedetection chambers 104.

When positive pressure is applied to the upstream side of the filter 120and/or negative pressure is applied to the downstream side of the filter120, a sample can be moved through the filter 120 to form a concentratedsample that is retained by the filter 120 (e.g., according to size,charge, and/or function) and a filtrate that passes through the filter120 and out of the sample processing system 100, and optionally, towaste or another receptacle. The sample processing system 100 canfurther include an aperture 131 (see FIG. 1) to which a port and/orvalve can be coupled. The aperture 131 can function as a vent when asample is introduced into the loading chamber 102 and/or when the sampleis filtered using the filters 120. Alternatively, or in addition, atleast a portion of the loading chamber 102 can deform and/or collapse asthe sample is filtered, for example, in response to negative pressure.

In some embodiments, the same process can be used to introduce a sampleinto the sample processing system 100 and to filter the sample. Forexample, in some embodiments, positive and/or negative pressure can beapplied across the loading chamber 102, for example, across the upstreamside of the aperture 111 and the downstream side of the filters 120,such that a sample is moved into the loading chamber 102 via theaperture 111, concentrated on the filters 120, and a filtrate of thesample is removed from the fluid path 110 of the sample processingsystem 100 via the filter outlets 124 (i.e., also functioning as thefiltrate outlets 126). As such, the sample can be introduced into thesample processing system 100 and concentrated in the sample processingsystem 100 in one simultaneous step, or in a plurality of sequentialsteps.

In some embodiments, the sample is filtered to completion (i.e.,substantially all liquid can be removed from the sample), and in someembodiments, the sample can be partially filtered, such that some liquidremains in the concentrated sample after filtration.

After the sample has been filtered using the filters 120 to form aconcentrated sample on each filter 120, one or more washing solutionscan optionally be introduced into the loading chamber 102 to wash theconcentrated sample on each filter 120. The introduction of the one ormore washing solutions can follow the same process as the initial sampleintroduction and/or filtering process.

Furthermore, an elution solution can be added to the loading chamber102, for example, following the same process as the initial sampleintroduction process. However, in general (and unlike washingsolutions), the elution solution will not be passed through the filters120. That is, the filter outlets 124 can be covered to close the filters120 to ambience when an elution solution is added to the loading chamber102. Alternatively, in some embodiments, the filters 120 can be removedand plugged or sealed with a similarly-sized object. The elutionsolution can include the same diluent that the sample comprised or adifferent material. The elution solution can be selected to elute all ora portion of the concentrated sample (i.e., one or more analytes ofinterest) from the filters 120 and/or to inhibit the elution of othersportions of the concentrated sample (i.e., one or more non-targetanalytes that may be present in the sample). In some embodiments, thevolume of elution solution added to the sample processing system 100 canbe controlled such that all of the volume is moved into the detectionchambers 104 following elution.

Optionally, the sample processing system 100 can be agitated tofacilitate the elution of all or a portion of the concentrated samplefrom the filters 120. In addition, in some embodiments, the sampleprocessing system 100 can be incubated to promote the growth of one ormore microorganisms of interest.

After all or a portion of the concentrated sample has been eluted fromone or more of the filters 120, all or a portion of the concentratedsample can be moved in the fluid path 110 (i.e., without exposing theconcentrated sample to ambience) to the detection chambers 104. That is,the concentrated sample (or a portion thereof) can be moved into one ormore of the plurality of primary channels 106, into one or more of thesecondary channels 108, and into one or more of the detection chambers104.

In some embodiments, the phrase “without exposing to ambience” andderivations thereof refers to not removing the sample and/orconcentrated sample from the sample processing system 100 during thetransfer between the loading chamber 102 and the detection chambers 104(e.g., to prevent spills or contamination, to facilitate samplehandling, to minimize sample loss, etc.), such that thesample/concentrated sample remains in the fluid path 110 of the sampleprocessing system 100 from sample introduction, concentration, elution,and transfer to the detection chambers 104. However, not being exposedto ambience does not necessarily mean that the sample processing system100 is closed to gas-exchange or that other liquids cannot be introducedinto the sample processing system 100. For example, in some embodiments,the loading chamber 102, one or more detection chambers 104, one or moreprimary channels 106, one or more secondary channels 108, a portionthereof, or a combination thereof, is gas-permeable, or includes agas-permeable film or membrane (e.g., to allow aerobic bacteria tocontinue to have access to oxygen). For example, in some embodiments, atleast one wall defining at least one of the detection chamber 104, thesecondary channel 108, the primary channel 106, and the loading chamber102 can be porous and can be adapted to allow gas exchange (e.g.,without being liquid-permeable).

The concentrated sample can be moved into detection chambers 104 in avariety of ways, including, but not limited to, employing a pressuredifferential, centrifuging, employing capillary action (e.g., wicking),controlling the surface energy of any surface in the fluid path 110between the filter 120 and the detection chamber 104, etc., or acombination thereof.

Employing a pressure differential can include applying positive pressureto fill the detection chambers 104 and/or vacuum filling the detectionchambers 104. For example, any of the above described means for applyingpositive pressure can be employed, including, but not limited to, usingan upstream manual and/or mechanical pump, employing a deformableloading chamber 102 (e.g., bulb-like), or combinations thereof. Vacuumfilling of the detection chambers 104 can be accomplished, for example,if the detection chambers 104 are gas-permeable and liquid-impermeable,such that a vacuum source can be applied to the second major side 105 ofthe sample processing system 100 adjacent the back side of the detectionchambers 104 to move the concentrated sample from the filters 120 to thedetection chambers 104. Such gas-permeability can also be employed tovent the detection chambers 104 to facilitate filling under positivepressure.

Centrifuging can be employed particularly in embodiments in which theanalyte(s) of interest represent the more dense matter in theconcentrated sample, such that the concentrated sample can be separatedby density. Centrifuging can include placing the sample processingsystem 100 in a centrifuge to facilitate moving the concentrated samplein the desired direction. For example, the sample processing system 100can be oriented such that the most dense matter will be directed intothe detection chambers 104 during centrifugation.

In the centrifugation step, the centrifugation g-force and/or durationnecessary to move the concentrated sample into the detection chambers104 can depending on one or more of the composition of the concentratesample, the analyte(s) of interest, the shape, dimensions and surfaceenergy of the filters 120, the surface energy of the concentratedsample, and the like. In some embodiments, the centrifugation can beperformed according to the processes described in Bedingham, et al.,U.S. Pat. No. 6,627,159, entitled “Centrifugal filling of sampleprocessing devices,” which is incorporated herein by reference. In someembodiments, it may be desired to fill substantially all of thedetection chambers 104 of the sample processing system 100 with theconcentrated sample (e.g., in enumeration assays).

In some embodiments, it may be desired to concentrate substantially allof the concentrated sample in one or more terminal detection chambers104 (e.g., in presence/absence assays). In such embodiments, the g-forceand/or duration necessary to move all of the concentrated sample to aterminal position of the sample processing system 100 can depend on thesize and density of the analyte, the density and viscosity of thediluent (e.g., the elution solution used), the volume of concentratedsample, and/or the distance the concentrated sample will be required totravel to reach the detection chambers 104 (e.g., the length of theprimary channels 106 and secondary channels 108). The sedimentationvelocity (V, in centimeters per second (cm/s)) can be approximated usingthe formula:

V=2ga ²(ρ1−ρ2)/9η

where g=acceleration in cm/s² (i.e., g-force in gs*980 cm/s²),ρ1=analyte density in g/cm³, ρ2=density of media (e.g., diluent, elutionsolution, etc.) in g/cm³, η=coefficient of viscosity in poises (g/cm/s),and a=analyte radius in centimeters (assuming a spherical shape). Insome centrifuges (e.g., some laboratory centrifuges), the g-force can bedetermined by the rotational speed (e.g., in revolutions per minute(RPM)) and the distance of the sample from the center of the rotor (i.e.the sample experiences a higher g-force at the same rotational speed ifit is placed further away from the rotor). The sedimentation velocitycan be calculated using the above equation, and then the centrifugationtime (i.e., duration) can be calculated by dividing the distance theconcentrated sample needs to travel by the sedimentation velocity.Alternatively, the desired time and distance can be used to estimate asedimentation velocity, and the necessary g-force can then be calculatedusing the above equation.

In some embodiments, the g-force in the centrifugation step can be atleast about 50 g (e.g., 50*9.8 m/s² on earth, at sea level), in someembodiments, at least about 500 g, and in some embodiments, at leastabout 5000 g. In some embodiments, the g-force in the centrifugationstep can be no greater than about 20,000 g, in some embodiments, nogreater than about 10,000 g, and in some embodiments, no greater thanabout 7500 g.

In some embodiments, the duration of the centrifugation step can be atleast about 10 seconds, in some embodiments, at least about 1 minutes,and in some embodiments, at least about 2 minutes. In some embodiments,the duration of the centrifugation step can be no greater than about 60minutes, in some embodiments, no greater than about 30 minutes, and insome embodiments, no greater than about 10 minutes.

As mentioned above, in some embodiments, capillary action (e.g.,wicking) can be used to move the concentrated sample into the detectionchambers 104. For example, one or more of the primary channels 106 andthe secondary channels 108 can be adapted to facilitate moving theconcentrated sample by capillary action from the loading chamber 102into the detection chambers 104. In some embodiments, one or more of theprimary channels 106 and the secondary channels 108 can include aplurality of microchannels to further facilitate wicking theconcentrated sample into the detection chambers 104.

Furthermore, in some embodiments, the surface energy of any of thesurfaces defining the fluid path 110 can be controlled to facilitatemoving the concentrated sample from the filters 120 to the detectionchambers 104. For example, in some embodiments, one or more surfaces ofone or more of the primary channels 106 can be modified (e.g., with ahydrophilic coating or surface treatment) to facilitate movement of theconcentrated sample (e.g., an aqueous concentrated sample) along therespective primary channels 106 into the detection chambers 104. Inaddition, or alternatively, in some embodiments, one or more surfacesdefining the fluid path 110 can be modified to facilitate movement ofthe concentrated sample off of the respective surface and toward anotherarea. For example, in some embodiments, the loading chamber 102 can besurface modified (e.g., with a hydrophobic coating or surface treatment)to facilitate movement of the concentrated sample (e.g., an aqueousconcentrated sample) away from the surfaces of the loading chamber 102and toward other areas, such as the primary channels 106.

After the concentrated sample has been moved into the detection chambers104, the concentrated sample can be inhibited from moving out of thedetection chambers 104, which can be accomplished in a variety of ways,including, but not limited to, obstructing or sealing at least a portionof the fluid path 110 upstream of the detection chambers 104, employingaspect ratios that minimize diffusion of the concentrated sample out ofthe detection chambers 104, etc., or a combination thereof.

In some embodiments, one or more of the primary channels 106 and thesecondary channels 108 can be obstructed and/or sealed to inhibit theconcentrated sample from moving out of the detection chambers 104 and/orback upstream of the detection chambers 104 using one or more ofadhesives, melt bonding, folding, crimping, etc., and combinationsthereof. For example, the sealing methods and systems described inBedingham, et al., PCT Publication No. WO 02/01180, which isincorporated herein by reference, can be employed in the presentdisclosure. Such sealing methods can be employed, for example, inembodiments such as the sample processing system 100 illustrated inFIGS. 1-3, in which the primary channels 106 are substantially parallelto one another along their length, and in which the secondary channels108 are relatively short, such that sealing at least the most downstreamportions of the primary channels 106 (e.g., simultaneously, asfacilitated by their parallel arrangement) can effectively inhibitmovement of the concentrated sample out of the detection chambers 104.

In some embodiments, the concentrated sample can be inhibited frommoving out of the detection chambers 104 by exploiting capillary forcesin the sample processing system 100 and/or by exploiting the surfacetension of the concentrated sample. That is, the concentrated sample canbe inhibited from moving out of a particular detection chamber 104 byensuring that any portion of the fluid path 110 (e.g., the secondarychannel 108 (or the primary channel 106 in embodiments in which asecondary channel 108 is not employed)) that is fluidly connected to thedetection chamber 104 has an aspect ratio relative to the detectionchamber 104 that minimizes diffusion (fluid movement) from the detectionchamber 104 into an upstream portion of the fluid path 110. That is, therelationship between the cross-sectional area of the fluid path 110(A_(p)) (e.g., at the outlet of the secondary channel 108) and thevolume (V) of the detection chamber 104 from which fluid may move intothe fluid path 110 can be controlled to inhibit diffusion from thedetection chamber 104 into an upstream portion of the fluid path 110. Inaddition, or alternatively, the aspect ratio between a primary channel106 and a particular secondary channel 108 can be controlled to inhibitmovement of the concentrated sample upstream into a primary channel 106.

For example, in some embodiments, the ratio of the cross-sectional areaof the fluid path 110 (A_(p)) (e.g., at the outlet of the secondarychannel 108) to the volume (V) of the detection chamber 104 from whichfluid may move into the fluid path 110, i.e., A_(p):V, can range fromabout 1:25 to about 1:500, in some embodiments, can range from about1:50 to about 1:300, and in some embodiments, can range from about 1:100to about 1:200. Said another way, in some embodiments, the fraction ofA_(p)/V can be at least about 0.01, in some embodiments, at least about0.02, and in some embodiments, at least about 0.04. In some embodiments,the fraction of A_(p)/V can be no greater than about 0.005, in someembodiments, no greater than about 0.003, and in some embodiments, nogreater than about 0.002. Reported in yet another way, in someembodiments, the fraction of V/A_(p), or the ratio of V to A_(p), can beat least about 25 (i.e., 25 to 1), in some embodiments, at least about50 (i.e., about 50 to 1), and in some embodiments, at least about 100(i.e., about 100 to 1). In some embodiments, the fraction of V/A_(p), orthe ratio of V to A_(p), can be no greater than about 500 (i.e., about500 to 1), in some embodiments, no greater than about 300 (i.e., about300 to 1), and in some embodiments, no greater than about 200 (i.e.,about 200 to 1).

In use, a liquid composition can be pre-filtered and/or pre-settled toform a liquid sample. Alternatively, a liquid sample can be used itselfwithout requiring any pre-filtering or pre-settling process. The samplecan be introduced into the loading chamber 102, for example, via theaperture 111 in the loading chamber 102. The sample can then be filteredwith the filters 120 to form a concentrated sample on the filters 120,while the filtrate is removed from the fluid path 110 of the sampleprocessing system 100, and is sent to waste or another receptacle. Afterthe concentrated sample has been formed on the filters 120 (e.g., bysize-restriction), one or more wash solutions can optionally beintroduced into the sample processing system 100 following the sameprocess as used for sample introduction and removed from the sampleprocessing system 100 following the sample concentration process.Following any washing steps employed, an elution solution can beintroduced into the sample processing system 100. The elution solutioncan be adapted to elute the concentrated sample from the filters 120,and can be adapted to selectively elute the portions of interest of theconcentrated sample from the filters 120. The sample processing system100 can be agitated to facilitate eluting the concentrated sample fromthe filters 120, such that a liquid is formed in the loading chamber 102comprising the elution solution and at least a portion of theconcentrated sample. The concentrated sample (and any elution solution)can then be moved into the detection chamber 104 using any of themethods described above. After the concentrated sample has been movedinto the detection chambers 104, the concentrated sample can beinhibited from moving out of the detection chambers 104, or upstream ofthe detection chambers 104 by any of the methods described above.

When the portions of interest of the concentrated sample have been movedinto (and retained) in the detection chambers 104, the sample processingsystem 100 can be used to enrich the analyte(s) of interest, if present,in the concentrated sample. That is, the detection chambers 104 caninclude one or more reagents, such as enrichment media. The reagents canbe provided in the detection chambers 104 in liquid or solid (e.g., drypowder) form, can be adsorbed or coated onto an inner surface of thedetection chambers 104, or a combination thereof. Enrichment media caninclude any media necessary to grow (e.g., in cell population) anyanalyte(s) of interest and/or to suppress the growth of any non-targetanalytes, and can include any of the materials listed above with respectto the diluent. Furthermore, the detection chambers 104 can include anyof a variety of indicators, such as color-change indicators, fluorescentindicators, chemiluminescent indicators, etc., and combinations thereof,in order to facilitate elucidating at least one of the presence,quantity and/or viability of the analyte(s) of interest.

In addition, or alternatively, in some embodiments, the sample can beenriched in the loading chamber 102 (e.g., in the presence of enrichmentmedia and at appropriate enrichment conditions, such as time,temperature, pressure, humidity, etc.), for example, to permit one ormore doubling cycles of bacteria of interest. After sufficientenrichment has occurred, the enriched sample can be concentrated (e.g.,using the filter(s) 120), and the concentrated sample can be eluted intothe detection chambers 104. In addition, or alternatively, enrichmentmedia can be added to the loading chamber 102 after the sample has beenconcentrated, and the concentrated sample can be enriched (andoptionally, refiltered) in the loading chamber 102 prior to being movedinto the detection chambers 104.

Thus, a sample can be introduced, concentrated and/or analyzed using thesample processing system 100, without ever ‘re-opening’ the sampleprocessing system 100 after the sample is first introduced into thesample processing system 100. As a result, the sample processing system100 can be a fully integrated sample processing device, in which asample can be analyzed or interrogated while minimizing bothcontamination and sample loss.

The sample processing system 100 illustrated in FIGS. 1-3 is shown byway of example only, however, it should be understood that a variety ofalternative shapes, numbers and configurations of the components of thesample processing system 100 can be employed without departing from thespirit and scope of the present disclosure. In addition, a variety ofconfigurations, features and/or processes described in Bedingham et al.‘Sample Processing’ patents and publications can be employed in thepresent disclosure, namely, U.S. Pat. Nos. 6,627,159, 6,720,187,6,734,401, 6,814,935, 6,869,666, 6,987,253, 7,026,168, 7,164,107,7,435,933, 7,445,752; US Patent Application Publication Nos.2004/179974, 2006/188396, 2006/189000, 2006/228811, 2006/269451; and PCTPatent Application Publication Nos. WO 02/00347, 02/01180, 02/01181,02/086454, 02/090091, and 2004/058405, all of which are incorporatedherein by reference.

For example, the loading chamber 102 is shaped to facilitate moving theconcentrated sample into the primary channels 106 (e.g., bycentrifugation), but it should be understood that any desired shape orconfiguration can be employed.

Furthermore, the primary channels 106 are illustrated as beingsubstantially parallel, which can facilitate moving the concentratedsample into the detection chambers and can also facilitate sealing theprimary channels simultaneously, but other shapes and arrangements ofthe primary channels 106 are possible.

Moreover, in embodiment shown in FIGS. 1-3, the primary channels 106,the secondary channels 108 and the detection chambers 104 are arrangedin arrays, such that each array includes forty-eight detection chambers104, forty-eight secondary channels 108, and one primary channel 106;there are eight arrays fluidly coupled to the loading chamber 102; andthere are four filters 120 employed in the loading chamber 102. However,a variety of numbers and arrangements of detection chambers 104,secondary channels 108, primary channels 106, filters 120 and loadingchambers 102 can be employed. For example, a variety of numbers andarrangements of detection chambers 104, secondary channels 108 andprimary channels 106 can be employed in each array, and as few arrays asone or as many as structurally possible can be employed in the sampleprocessing system 100. In addition, multiple loading chambers 102 can beemployed, such that there is one array fluidly coupled to each loadingchamber 102 (such as the embodiment illustrated in FIGS. 4-5, describedbelow). For example, in some embodiments, the sample processing system100 can include one loading chamber 102, one filter 120, one primarychannel 106, and one detection chamber 104.

In addition, some embodiments do not include any secondary channels 108,and in some embodiments, each secondary channel 108 can be an aperturebetween the detection chamber 104 and a respective primary channel 106,providing fluid communication between the detection chamber and theprimary channel 106.

Furthermore, in the embodiment illustrated in FIGS. 1-3, the sampleprocessing system 100 is shown as including one large loading chamber102 with multiple filters 120. However, in some embodiments, the onelarge loading chamber 102 can instead include one large filter 120(e.g., sized and/or shaped corresponding to the loading chamber 102). Insome embodiments, the sample processing system 100 can include as few oras many filters 120 as structurally possible.

Moreover, in the embodiment illustrated in FIGS. 1-3, the secondarychannels 108 are shown in pairs, exiting off of the respective primarychannel 106 at the same downstream location. However, a variety of otherconfigurations of secondary channels 108 can be employed, such asoff-set locations, etc., as further described in the above-mentionedBedingham et al. patents and patent publications.

In addition, the sample processing system 100 can be formed in a varietyof ways, such as the processes described in the above-mentionedBedingham et al. patents and publications.

FIGS. 4-5 illustrate a sample processing system 200 according to anotherembodiment of the present disclosure. The sample processing system 200shares many of the same elements and features described above withreference to the illustrated embodiment of FIGS. 1-3. Accordingly,elements and features corresponding to elements and features in theillustrated embodiments of FIGS. 1-3 are provided with the samereference numerals in the 200 series. Reference is made to thedescription above accompanying FIGS. 1-3 for a more complete descriptionof the features and elements (and alternatives to such features andelements) of the embodiment illustrated in FIGS. 4-5.

The sample processing system 200 includes a first major side 203, asecond major side 205, and a plurality of sample processing arrays 201(eight are shown in FIG. 4 by way of example only). By way of exampleonly, each sample processing array 201 is illustrated in FIG. 4 asincluding one loading chamber 202, a plurality of detection chambers 204(forty-eight are shown in FIG. 4 by way of example only), one primarychannel 206, and a plurality of secondary channels 208 (forty-eight,i.e., one per detection chamber 104, are shown in FIG. 4 by way ofexample only) positioned to fluidly couple the plurality of detectionchambers 204 to the primary channel 206.

Each sample processing array 201 can define a fluid path 210, such thatthe sample processing system 200 includes a plurality of fluid paths 210that, in some embodiments, can be fluidly isolated from one another.Each fluid path 210 is at least partially defined by the respectivearray's loading chamber 202, plurality of detection chambers 204,primary channel 206, and plurality of secondary channels 208.

The same sample, or a plurality of samples, can be concentrated andanalyzed using the sample processing system 200. For example, in someembodiments, the sample processing system 200 can be used tosimultaneously process a variety of samples in parallel. Samples can beintroduced into the sample processing system 200 using apertures similarto the aperture 111 and any ports or valves coupled thereto, asillustrated in FIG. 1 and described above. Additionally, oralternatively, in some embodiments, samples can be introduced into theloading chambers 202 by puncturing a wall defining the loading chamber202 (e.g., with a needle and syringe). In addition, if necessary,introduction of the sample can be facilitated by venting the loadingchamber 202 as the sample is introduced. For example, an aperturesimilar to the aperture 131 illustrated in FIG. 1 as described above canbe employed. Additionally, or alternatively, in some embodiments, a walldefining the loading chamber 202 can be punctured to vent the loadingchamber 202. For example, as described in the Bedingham et al. patentsand publications, a first aperture can be created (e.g., in a rear wall235) in one portion of the loading chamber 202, and a second aperturecan be created in another location (e.g., in the other rear wall 237 ofthe generally U-shaped loading chamber 202) to introduce the sample intothe loading chamber 202. The U-shaped loading chambers 202 are shown inFIGS. 4-5 by way of example only, however, it should be understood thata variety of shapes and configurations can be employed for the loadingchambers 202.

As shown in FIGS. 4-5, each sample processing array 201 can furtherinclude one or more filters 220 positioned in the loading chamber 202.Each filter 220 can include an inlet 222 and an outlet 224, and eachfilter 220 can be positioned such that at least one of the inlet 222 andthe outlet 224 is positioned in the fluid path 210. Similar to theembodiment illustrated in FIGS. 1-3, the filter inlet 222 of each filter220 illustrated in FIGS. 4-5 is positioned in the fluid path 210, andthe outlet 224 of each filter 220 is positioned such that any filtrateformed by the filter 220 is removed from the fluid path 210 via thefilter outlet 224. As a result, each filter 220 defines a filter fluidpath 225 (see FIG. 5). As such, the fluid path 210 can be referred to asa “primary fluid path” of the system 200, or the respective array 201,and the filter fluid path 225 can be referred to as a “secondary fluidpath,” and in some embodiments, the secondary fluid path 225 can beoriented at an angle with respect to the primary fluid path 210. Forexample, as shown in FIG. 5, the fluid path 210 is configured such thatfluid can flow in the fluid path 210 generally along a first directionD₁, and the filter 220 is arranged such that fluid can flow through thefilter 220 in the filter fluid path 225 generally along a seconddirection D₂, and by way of example only, the first direction D₁ and thesecond direction D₂ are shown as being oriented substantiallyperpendicularly with respect to one another, although other angles oforientation are possible.

Each filter 220 can be adapted to form a concentrated sample and afiltrate. Each sample processing array 201 can include an outlet 226,i.e., a filtrate outlet, for removing the filtrate of the sample, or theundesired portions of the sample, from the fluid path 210, andparticularly, for removing the filtrate from the fluid path 210 at alocation upstream of the detection chambers 204. In some embodiments, asshown in FIGS. 4-5, each filter 220 is positioned such that the filteroutlet 224 is directed out of the fluid path 210, and, as a result, thefiltrate outlet 226 includes the filter outlet 224. That is, in theembodiment in FIGS. 4-5, each filter 220 is positioned such thatportions of a sample flowing through the filter 220 (i.e., the filtrate)will be directed out of the fluid path 210, and particularly, away fromthe detection chambers 204.

In use, the sample processing system 200, and each sample processingarray 201 can be used to process a sample similarly to the methods, andalternatives thereto, described above with respect to the sampleprocessing system 100 illustrated in FIGS. 1-3.

FIG. 6 illustrates a sample processing system 300 according to anotherembodiment of the present disclosure. The sample processing system 300shares many of the same elements and features described above withreference to the illustrated embodiments of FIGS. 1-5. Accordingly,elements and features corresponding to elements and features in theillustrated embodiments of FIGS. 1-5 are provided with the samereference numerals in the 300 series. Reference is made to thedescription above accompanying FIGS. 1-5 for a more complete descriptionof the features and elements (and alternatives to such features andelements) of the embodiment illustrated in FIG. 6.

The sample processing system 300 includes a first major side 303 and asecond major side 305. The sample processing system 300 further includesone or more loading chamber 302, one or more detection chambers 304, oneor more primary channels 306, and one or more secondary channels 308.The sample processing system 300 further includes a fluid path 310 thatis at least partially defined by the one or more loading chambers 302,the one or more detection chambers 304, the one or more primary channels306, and the one or more secondary channels 308 (if employed). Forsimplicity, the sample processing system 300 will be described, withreference to FIG. 6, as including one loading chamber 302, one detectionchamber 304, one primary channel 306, and one secondary channel 308, butit should be understood that the various arrangements and configurationsdescribed above with respect to the sample processing system 100illustrated in FIGS. 1-3 can also be employed in the sample processingsystem 300 illustrated in FIG. 6

As shown in FIG. 6, the sample processing system 300 can further includeone or more filters 320 positioned in the loading chamber 302. Thefilter 320 can include an inlet 322 and an outlet 324, and the filter320 is shown as being positioned such that both the inlet 322 and theoutlet 324 are positioned in the fluid path 310. As a result, the filter320 defines a filter fluid path 325. As such, the fluid path 310 can bereferred to as a “primary fluid path” of the system 300, and the filterfluid path 325 can be referred to as a “secondary fluid path” In theembodiment illustrated in FIG. 6, the secondary fluid path 325 is inline with the primary fluid path 310, such that fluid can flow in thefluid path 310 generally along a first direction D₁, and the filter 320is arranged such that fluid can also flow through the filter 320generally along the first direction D₁.

In some embodiments employing a filter fluid path 325 that is generallyin line with the fluid path 210, the filter 320 can be configured, forexample, to filter the sample by charge- and/or function-filtration. Assuch, the filter 320 can be configured to retain (e.g., temporarily) theportions of the sample of interest (e.g., the portions including theanalyte(s) of interest), while the remainder of the sample (i.e., thefiltrate) passes through the filter 320.

In such embodiments, the sample processing system 300 can include afiltrate outlet 326 positioned downstream of the filter 320 but upstreamof the detection chamber 304 such that any fluid moved through thefilter 320 can be removed from the fluid path 310 before being movedinto the detection chambers 304. For example, the filtrate outlet 326can allow fluid to flow out of the fluid path 310 and generally along asecond direction D₂, which can be oriented at an angle with respect tothe first direction D₁. By way of example only, the first direction D₁and the second direction D₂ can be oriented substantiallyperpendicularly with respect to one another, although other angles oforientation are possible.

In some embodiments, as shown in FIG. 6, at least a portion of the fluidpath 310 (e.g., a portion of the fluid path 310 that is positioneddownstream of the filter 320 and upstream of the detection chamber 304)can be adapted to change between a first, closed state in which thefilter 320 and the detection chambers are not in fluid communication anda second, open state in which the filter 320 and the detection chamber304 are in fluid communication. As a result, the fluid path 310 can bein the first, closed state during filtration and removal of the filtrate(i.e., during formation of the concentrated sample), and the fluid path310 can be in the second, open state during movement of the concentratedsample to the detection chamber 304.

Accordingly, in some embodiments, the sample processing system caninclude a member (obstruction, seal, etc.) 340 positioned to obstructthe fluid path 310 at a position that is located downstream of thefilter outlet 324 and upstream of the detection chamber 304. In someembodiments, the member 340 can be movable between a first position P₁in which the member is obstructing the fluid path 310 (e.g., in whichthe member 340 is causing the fluid path 310 to be in its first, closedstate) and a second position P₂ in which the member 340 is notobstructing the fluid path 310 (e.g., in which the member 340 is causingthe fluid path 310 to be in its second, open state).

As a result, the member 340 can be positioned in the first position P₁during filtration and removal of the filtrate via the filtrate outlet326 (i.e., during formation of the concentrated sample), and the member340 can be positioned in the second position P₂ during movement of theconcentrated sample to the detection chamber 304.

In some embodiments, additionally, or alternatively, selectivelyactuatable valves can be positioned in the fluid path 310 to controlfluid movement between the filter 320 and the detection chamber 304.

Such constructions of the fluid path 310 (e.g., employing one or more ofmovable member(s) 340, valves, etc.) can be useful, for example, inembodiments in which positive pressure is used to move the samplethrough the filter 320. In embodiments employing negative pressure, forexample, such constructions may not be necessary, as the entire fluidpath 310 can be evacuated during filtration of the sample.

The filtrate outlet 326 can be also adapted to change between a first,open state in which the downstream side of the filter 320 (i.e., theoutlet 324) is in fluid communication with ambience (or anotherdownstream system or process) via the filtrate outlet 326, and a second,closed state in which the downstream side of the filter 320 (i.e., theoutlet 324) is not in fluid communication with ambience or anotherdevice, via the filtrate outlet 326. Constructions such as thosedescribed above (e.g., movable member(s), valves, etc.) can be used tocontrol fluid movement through the filtrate outlet 326, when necessary.

In use, the sample processing system 300 can be used to process a samplesimilarly to the methods, and alternatives thereto, described above withrespect to the sample processing system 100 illustrated in FIGS. 1-3.

After the sample has been concentrated on the filter 320, the filter 320and concentrated sample can be washed with one or more washing solutions(and one or more steps) following the same process described above,wherein the washing solution can be removed from the fluid path 310 viathe filtrate outlet 326. Following the optional washing step(s), anelution solution can be added to the loading chamber 302 following thesame procedure as was used to introduce a sample to the sampleprocessing system 300. Such an elution solution can be adapted todisrupt any interaction between the filter 320 and the analyte(s) ofinterest (or the portions of interest in the concentrated sample), thefiltrate outlet 326 can be changed to its second, closed state, themember 340 can be moved into its second position P₂, and theconcentrated sample (or the portions of interest) can be moved in thefluid path 310 to the detection chamber 304.

While the sample processing systems 100, 200 and 300 are illustrated anddescribed separately above, it should be understood that any combinationof the above-described sample processing systems 100, 200, and 300 andmethods are possible and within the spirit and scope of the presentdisclosure.

As described above, a variety of filters 120, 220, 320 can be employedin the sample processing system 100, 200, 300 of the present disclosureto concentrate a sample by size, charge and/or function. For example,filters 120, 220, 320 can be employed that carry a surface charge forthe ionic capture of soluble analytes. By way of example only, such asurface charge may be useful in embodiment in which bacteria undergo alysis step, for example, prior to concentration and/or movement into thedetection chambers 104. For example, in some embodiments, nucleic acidcan be captured on a filter 120, 220, 320 comprising glass fiber,followed by washing and elution into the detection chambers 104.

Furthermore, the sample processing systems 100, 200, 300 can be usefulin assaying a variety of analyte(s) of interest. For example, any of thesample processing systems 100, 200, 300 disclosed herein can be used ininfectious disease diagnostics where a low number of bacteria may bepresent in the original sample. Bacterial concentration can be achievedusing one or more filters 120, 220, 320, followed by cell lysis,optional DNA purification, and finally elution into the detectionchambers 104 that can contain reagents for identifying the bacteria(e.g., polymerase chain reaction primers and probes, etc.).

The following working examples are intended to be illustrative of thepresent disclosure and not limiting.

EXAMPLES Example 1 Bacterial Enumeration Assay Sample Processing System

Four sample processing systems 100 were assembled according to theembodiment illustrated in FIGS. 1-3, the four systems serving as fourreplicates in this example. The sample processing systems 100 eachincluded a single loading chamber 102, 8 primary channels 106, and 384secondary channels 108 and detection chambers 104, each primary channel106 providing fluid communication between the loading chamber 102 and 48secondary channels 108 and detection chambers 104. A first Luer port wascoupled to the aperture 111, and a second Luer port was coupled to theaperture 131 of each sample processing system. Each sample processingsystem 100 further included four filters 120 positioned in the loadingchamber 102, and each filter 120 was 10 mm in diameter and punched froma METRICEL® membrane disc filter (0.45-micrometer pore size, availablefrom Pall Corporation, East Hills, N.Y.).

The first major side 103 was formed of polypropylene, and the loadingchamber 102, primary channels 106, secondary channels 108 and detectionchambers 104 were form by thermoforming. The second major side 105 wasformed of an aluminum foil coated with a silicone-polyurea adhesive, asgenerally described in Bedingham, et al., U.S. Pat. No. 7,026,168.

The sample processing systems 100 were assembled according to themethods described in Bedingham et al., PCT Application Publication No.WO 02/01180, with additional processing steps for the addition of thefilters 120, apertures 111, 131 and corresponding Luer ports, etc.

To form each sample processing system replicate, the assemblies wereplaced in a machined tool designed to mate with the embossed pattern inthe first major side 103 while fully contacting the flat land areabetween embossed features. The assemblies and tools were then placed ina laminating press (Model 810, available from Sencorp, Inc., Hyannis,Mass.) at 200° F. for 5 seconds at 60 psi to bond the first major side103 to the second major side 105.

Filtration Process

A vacuum manifold designed to mate to the second major side 105 of eachsample processing system replicate under the loading chamber 102 wasassembled and coupled to a vacuum flask, which was, in turn, coupled toa vacuum source. To ensure sealing between the manifold and the device,a rubber gasket was cut to size and placed between the manifold and thesample processing system 100. To support the rear side of the filters120 during filtration, a layer of a semi-rigid nonwovern material(CELESTRA® spunbonded polypropylene, available from Fiberweb Inc.,London, England) was placed between the gasket and the manifold. A pieceof silicone tubing approximately 8″ in length having a ⅛-inch internaldiameter was cut and coupled to one of the Luer ports using a barbedadapter to serve as the connector 109.

Sterile H₂O was introduced into each sample processing system via theconnector 109 using a 3-cc syringe to fill the loading chamber 102 andto wet the filters 120. The syringe was removed and the connector 109was then placed into 100 mL of an approximately 1 cfu/mL sample of E.coli (ATCC MG1655) prepared in Butterfield's Buffer from an overnightculture of approximately 10⁹ organisms/mL grown in BBL™ Trypticase SoyBroth (available from Becton, Dickinson and Co., Franklin Lakes, N.J.).The sample was filtered through the filters 120, capturing the bacteriaand allowing the buffer solution to flow out of the fluid path 110 ofthe respective sample processing system 100 and into the vacuum flask.

Elution and Movement into Detection Chambers

A stock solution of 4-methylumbelliferyl phosphate (MU-phos) in LuriaBroth (LB) having a concentration of 250 μg/mL was prepared by adding15.5 g of LB to 1000 mL of sterile water in a Pyrex bottle. The bottlewas partially sealed and autoclaved to sterilize the solution. Oncesufficiently cooled, 250 mg of MU-phos was added to the LB to make thestock solution. The solution was swirled vigorously to ensuredissolution of the indicator.

After filtering the sample using the sample processing systems 100 toconcentrate the sample, the stopper from one Luer port was opened tocreate a vent. 1 mL of the above-described stock solution of MU-phos inLB was introduced to the loading chamber 102 through the second Luerport using a 1-cc syringe. The backside of the filters 120 were coveredusing autoclave tape (3M™ ESPE™ autoclave steam indicator, availablefrom 3M Company, St. Paul, Minn.). Both ports were then sealed usingLuer caps. To release captured bacteria from the filters 120, the devicewas placed on a vortex mixer with a flat platform (a VWR® Mini Vortexer,speed setting 6, available from VWR International, West Chester, Pa.)for 30 seconds. The sample processing system replicates were thencentrifuged according to the process outlined in Bedingham, et al., U.S.Pat. No. 6,627,159 (i.e., at 2000 RPM for 2 min) to move the elutedconcentrated sample into the detection chambers 104 via the primarychannels 106. The primary channels 106 were then sealed using a stylusdevice, as described in Bedingham, et al., PCT Application PublicationNo. WO 02/01180. Each sample processing system replicate was thenincubated for 18 hours at 37° C.

Fluorescene Imaging of Detection Chambers

After incubation, each sample processing system was imaged on a gelreader (Alpha Innotech Chemimager model 5500, available from AlphaInnotech, San Leandro, Calif.) using overhead illumination (365 nm) anda UV cutoff filter. Images were acquired using 8-second exposure time.FIGS. 7-10 show the four images of the four replicate sample processingsystems tested using the procedures outlined above, where FIG. 7illustrates Replicate 1 of Table 1, FIG. 8 illustrates Replicate 2 ofTable 1, FIG. 9 illustrates Replicate 3 of Table 1, and FIG. 10illustrates Replicate 4 of Table 1. Additionally, the systems wereplaced in a fluorescence plate reader (available under the tradedesignation “SPECTRAMAX,” available from Molecular Devices, Sunnyvale,Calif.). The fluorescence intensity of each detection chamber 104 wasdetermined using 350 nm excitation and 450 nm emission. In this manner,an “automated” readout was achieved.

Enumeration Using Most Probable Number

The formula MPN=N ln(N/N−X) was used to estimate the most probablenumber, where N is the total number of detection chambers and X is thenumber of “positive” compartments exhibiting fluorescence. Since thedetection chambers did not contain a full milliliter of volume, the MPNvalue was multiplied by 1.74 (384 1.5-μL detection chambers). Table 1shows the results from the four replicates that were tested using theprocedure outlined above. In each device, 100 mL of sample wasconcentrated followed by 18 hours of incubation. A sample of the 100/mLdilution used to prepare the final 1/mL dilutions was also plated on 3M™PETRIFILM™ EC plates (available from 3M Company, St. Paul, Minn.),indicating a count of 89 CFU/mL. Enumeration results calculated usingthe MPN formula for each replicate are shown in Table 1.

TABLE 1 ENUMERATION RESULTS FOR FOUR REPLICATES OF A SAMPLE PROCESSINGSYSTEM REPLICATE COUNT MPN RESULT 1 23 41 2 38 70 3 23 41 4 17 30

Example 2 Bacterial Enumeration Assay

Two replicates of a sample processing system were prepared according tothe procedure outlined above in Example 1, except that the filters 120were isoporous membranes (PORETICS® polycarbonate black membrane, 0.4micron pore size, available from GE Osmonics, Minnetonka, Minn.).Filtration (i.e., concentration), elution, detection, and enumerationwere performed as described above in Example 1. The ˜100 cfu/mL sampleused to prepare the final ˜1/mL dilution was plated on a 3M™ PETRIFILM™EC plate (available from 3M Company, St. Paul, Minn.), as a control. Theimages from two replicates are shown in FIGS. 11 and 12, where FIG. 11illustrates Replicate 1 of Table 2, and FIG. 12 illustrates Replicate 2of Table 2. Numerical results for the two replicates are shown in Table2.

TABLE 2 ENUMERATION RESULTS FOR TWO REPLICATES OF A SAMPLE PROCESSINGSYSTEM REPLICATE COUNT MPN RESULT CONTROL COUNT 1 23 40 24 2 9 16 21

The embodiments described above and illustrated in the figures arepresented by way of example only and are not intended as a limitationupon the concepts and principles of the present disclosure. As such, itwill be appreciated by one having ordinary skill in the art that variouschanges in the elements and their configuration and arrangement arepossible without departing from the spirit and scope of the presentdisclosure. Various features and aspects of the present disclosure areset forth in the following claims.

1. A method for processing samples, the method comprising: providing aloading chamber; providing a detection chamber positioned in fluidcommunication with the loading chamber; providing a fluid path definedat least partially by the loading chamber and the detection chamber;positioning a sample in the loading chamber; filtering the sample with afilter positioned in the loading chamber to form a concentrated sampleon the filter and a filtrate, the filter having an inlet and an outletand positioned such that at least one of the inlet and the outlet ispositioned in the fluid path, wherein filtering includes applying apressure differential across the filter; removing the filtrate from thefluid path at a location upstream of the detection chamber; moving atleast a portion of the concentrated sample from the filter in the fluidpath to the detection chamber by centrifugation; and analyzing the atleast a portion of the concentrated sample in the detection chamber foran analyte of interest. 2-3. (canceled)
 4. The method of claim 1,wherein the inlet of the filter is positioned in the fluid path and theoutlet is positioned such that removing the filtrate from the fluid pathincludes removing the filtrate via the outlet of the filter and occursas a result of filtering the sample.
 5. The method of claim 1, whereinthe inlet and the outlet of the filter are positioned in the fluid path,such that the filtrate is removed from the fluid path at a location thatis downstream of the filter. 6-7. (canceled)
 8. The method of claim 1,wherein the fluid path is a primary fluid path and the filter defines asecondary fluid path, and wherein the secondary fluid path is orientedat an angle with respect to the primary fluid path.
 9. The method ofclaim 1, further comprising eluting the concentrated sample from thefilter in the fluid path.
 10. The method of claim 1, wherein moving atleast a portion of the concentrated sample in the fluid path includeseluting at least a portion of the concentrated sample off of the filter.11-12. (canceled)
 13. The method of claim 9, further comprisingagitating the loading chamber to facilitate eluting the concentratedsample from the filter.
 14. (canceled)
 15. The method of claim 1,wherein providing a detection chamber includes providing a plurality ofdetection chambers positioned in fluid communication with the loadingchamber, and wherein moving at least a portion of the concentratedsample in the fluid path to the detection chamber includes moving atleast a portion of the concentrated sample in the fluid path to theplurality of detection chambers.
 16. (canceled)
 17. The method of claim15, wherein the fluid path is further defined by a plurality of channelspositioned to fluidly couple the plurality of detection chambers withthe loading chamber, and wherein moving at least a portion of theconcentrated sample from the filter, in the fluid path to the detectionchamber includes moving at least a portion of the concentrated samplealong the plurality of channels.
 18. The method of claim 15, wherein thefluid path is further defined by a primary channel positioned to providefluid communication between the loading chamber and the plurality ofdetection chambers, wherein the fluid path is further defined by aplurality of secondary channels positioned to provide fluidcommunication between the plurality of detection chambers and theprimary channel, and wherein moving at least a portion of theconcentrated sample from the filter, in the fluid path to the detectionchamber includes moving at least a portion of the concentrated samplealong at least a portion of the primary channel and at least onesecondary channel to at least one detection chamber.
 19. (canceled) 20.The method of claim 1, further comprising inhibiting the at least aportion of the concentrated sample in the detection chamber from movingout of the detection chamber.
 21. The method of claim 20, whereininhibiting the at least a portion of the concentrated sample in thedetection chamber from moving out of the detection chamber includesinhibiting by capillary forces.
 22. The method of claim 20, whereininhibiting the at least a portion of the concentrated sample in thedetection chamber from moving out of the detection chamber includespositioning at least one of an obstruction and a seal in the fluid pathupstream of the detection chamber.
 23. (canceled)
 24. The method ofclaim 1, further comprising sealing at least a portion of the fluid pathafter moving at least a portion of the concentrated sample in the fluidpath to the detection chamber. 25-32. (canceled)
 33. A system forprocessing samples, the system comprising: a loading chamber adapted toreceive a sample; a detection chamber positioned in fluid communicationwith the loading chamber; a fluid path defined at least partially by theloading chamber and the detection chamber; a filter positioned in theloading chamber and having an inlet and an outlet, the filter positionedsuch that at least one of the inlet and the outlet is positioned in thefluid path, the filter adapted to filter the sample to form aconcentrated sample and a filtrate; and a filtrate outlet positionedsuch that the filtrate is removed from the fluid path at a locationupstream of the detection chamber.
 34. The system of claim 33, whereinthe filtrate outlet includes the outlet of the filter.
 35. The system ofclaim 33, wherein the inlet and the outlet of the filter are positionedin the fluid path, and wherein the filtrate outlet is positioned at alocation that is downstream of the filter. 36-37. (canceled)
 38. Thesystem of claim 33, further comprising a channel positioned in fluidcommunication between the loading chamber and the detection chamber, thechannel further defining at least a portion of the fluid path.
 39. Thesystem of claim 38, wherein at least a portion of the channel includes asurface modification adapted to facilitate movement of the concentratedsample to the detection chamber.
 40. The system of claim 38, wherein thedetection chamber is one of a plurality of detection chambers positionedin fluid communication with the loading chamber, and wherein the channelis one of a plurality of channels positioned to fluidly couple theplurality of detection chambers with the loading chamber.
 41. The systemof claim 38, wherein the detection chamber is one of a plurality ofdetection chambers positioned in fluid communication with the loadingchamber, and wherein the channel is a primary channel, and furthercomprising a plurality of secondary channels positioned to fluidlycouple the plurality of detection chambers with the primary channel. 42.The system of claim 38, wherein the detection chamber is one of aplurality of detection chambers positioned in fluid communication withthe loading chamber, and wherein the loading chamber is one of aplurality of loading chambers, and wherein at least some of theplurality of detection chambers are positioned in fluid communicationwith at least one of the plurality of loading chambers. 43-47.(canceled)